SummaryThe formation of a functional patterned vascular network is essential for development, tissue growth and organ physiology. Several human vascular disorders arise from the mis-patterning of blood vessels, such as arteriovenous malformations, aneurysms and diabetic retinopathy. Although blood flow is recognised as a stimulus for vascular patterning, very little is known about the molecular mechanisms that regulate endothelial cell behaviour in response to flow and promote vascular patterning.
Recently, we uncovered that endothelial cells migrate extensively in the immature vascular network, and that endothelial cells polarise against the blood flow direction. Here, we put forward the hypothesis that vascular patterning is dependent on the polarisation and migration of endothelial cells against the flow direction, in a continuous flux of cells going from low-shear stress to high-shear stress regions. We will establish new reporter mouse lines to observe and manipulate endothelial polarity in vivo in order to investigate how polarisation and coordination of endothelial cells movements are orchestrated to generate vascular patterning. We will manipulate cell polarity using mouse models to understand the importance of cell polarisation in vascular patterning. Also, using a unique zebrafish line allowing analysis of endothelial cell polarity, we will perform a screen to identify novel regulators of vascular patterning. Finally, we will explore the hypothesis that defective flow-dependent endothelial polarisation underlies arteriovenous malformations using two genetic models.
This integrative approach, based on high-resolution imaging and unique experimental models, will provide a unifying model defining the cellular and molecular principles involved in vascular patterning. Given the physiological relevance of vascular patterning in health and disease, this research plan will set the basis for the development of novel clinical therapies targeting vascular disorders.

The formation of a functional patterned vascular network is essential for development, tissue growth and organ physiology. Several human vascular disorders arise from the mis-patterning of blood vessels, such as arteriovenous malformations, aneurysms and diabetic retinopathy. Although blood flow is recognised as a stimulus for vascular patterning, very little is known about the molecular mechanisms that regulate endothelial cell behaviour in response to flow and promote vascular patterning.
Recently, we uncovered that endothelial cells migrate extensively in the immature vascular network, and that endothelial cells polarise against the blood flow direction. Here, we put forward the hypothesis that vascular patterning is dependent on the polarisation and migration of endothelial cells against the flow direction, in a continuous flux of cells going from low-shear stress to high-shear stress regions. We will establish new reporter mouse lines to observe and manipulate endothelial polarity in vivo in order to investigate how polarisation and coordination of endothelial cells movements are orchestrated to generate vascular patterning. We will manipulate cell polarity using mouse models to understand the importance of cell polarisation in vascular patterning. Also, using a unique zebrafish line allowing analysis of endothelial cell polarity, we will perform a screen to identify novel regulators of vascular patterning. Finally, we will explore the hypothesis that defective flow-dependent endothelial polarisation underlies arteriovenous malformations using two genetic models.
This integrative approach, based on high-resolution imaging and unique experimental models, will provide a unifying model defining the cellular and molecular principles involved in vascular patterning. Given the physiological relevance of vascular patterning in health and disease, this research plan will set the basis for the development of novel clinical therapies targeting vascular disorders.

Max ERC Funding

1 618 750 €

Duration

Start date: 2016-09-01, End date: 2021-08-31

Project acronymBETATOBETA

ProjectThe molecular basis of pancreatic beta cell replication

Researcher (PI)Yuval Dor

Host Institution (HI)THE HEBREW UNIVERSITY OF JERUSALEM

Call DetailsStarting Grant (StG), LS4, ERC-2010-StG_20091118

SummaryA fundamental challenge of pancreas biology is to understand and manipulate the determinants of beta cell mass. The homeostatic maintenance of adult beta cell mass relies largely on replication of differentiated beta cells, but the triggers and signaling pathways involved remain poorly understood. Here I propose to investigate the physiological and molecular mechanisms that control beta cell replication. First, novel transgenic mouse tools will be used to isolate live replicating beta cells and to examine the genetic program of beta cell replication in vivo. Information gained will provide insights into the molecular biology of cell division in vivo. Additionally, these experiments will address critical unresolved questions in beta cell biology, for example whether duplication involves transient dedifferentiation. Second, genetic and pharmacologic tools will be used to dissect the signaling pathways controlling the entry of beta cells to the cell division cycle, with emphasis on the roles of glucose and insulin, the key physiological input and output of beta cells. The expected outcome of these studies is a detailed molecular understanding of the homeostatic maintenance of beta cell mass, describing how beta cell function is linked to beta cell number in vivo. This may suggest new targets and concepts for pharmacologic intervention, towards the development of regenerative therapy strategies in diabetes. More generally, the experiments will shed light on one of the greatest mysteries of developmental biology, namely how organs achieve and maintain their correct size. A fundamental challenge of pancreas biology is to understand and manipulate the determinants of beta cell mass. The homeostatic maintenance of adult beta cell mass relies largely on replication of differentiated beta cells, but the triggers and signaling pathways involved remain poorly understood. Here I propose to investigate the physiological and molecular mechanisms that control beta cell replication. First, novel transgenic mouse tools will be used to isolate live replicating beta cells and to examine the genetic program of beta cell replication in vivo. Information gained will provide insights into the molecular biology of cell division in vivo. Additionally, these experiments will address critical unresolved questions in beta cell biology, for example whether duplication involves transient dedifferentiation. Second, genetic and pharmacologic tools will be used to dissect the signaling pathways controlling the entry of beta cells to the cell division cycle, with emphasis on the roles of glucose and insulin, the key physiological input and output of beta cells. The expected outcome of these studies is a detailed molecular understanding of the homeostatic maintenance of beta cell mass, describing how beta cell function is linked to beta cell number in vivo. This may suggest new targets and concepts for pharmacologic intervention, towards the development of regenerative therapy strategies in diabetes. More generally, the experiments will shed light on one of the greatest mysteries of developmental biology, namely how organs achieve and maintain their correct size.

A fundamental challenge of pancreas biology is to understand and manipulate the determinants of beta cell mass. The homeostatic maintenance of adult beta cell mass relies largely on replication of differentiated beta cells, but the triggers and signaling pathways involved remain poorly understood. Here I propose to investigate the physiological and molecular mechanisms that control beta cell replication. First, novel transgenic mouse tools will be used to isolate live replicating beta cells and to examine the genetic program of beta cell replication in vivo. Information gained will provide insights into the molecular biology of cell division in vivo. Additionally, these experiments will address critical unresolved questions in beta cell biology, for example whether duplication involves transient dedifferentiation. Second, genetic and pharmacologic tools will be used to dissect the signaling pathways controlling the entry of beta cells to the cell division cycle, with emphasis on the roles of glucose and insulin, the key physiological input and output of beta cells. The expected outcome of these studies is a detailed molecular understanding of the homeostatic maintenance of beta cell mass, describing how beta cell function is linked to beta cell number in vivo. This may suggest new targets and concepts for pharmacologic intervention, towards the development of regenerative therapy strategies in diabetes. More generally, the experiments will shed light on one of the greatest mysteries of developmental biology, namely how organs achieve and maintain their correct size. A fundamental challenge of pancreas biology is to understand and manipulate the determinants of beta cell mass. The homeostatic maintenance of adult beta cell mass relies largely on replication of differentiated beta cells, but the triggers and signaling pathways involved remain poorly understood. Here I propose to investigate the physiological and molecular mechanisms that control beta cell replication. First, novel transgenic mouse tools will be used to isolate live replicating beta cells and to examine the genetic program of beta cell replication in vivo. Information gained will provide insights into the molecular biology of cell division in vivo. Additionally, these experiments will address critical unresolved questions in beta cell biology, for example whether duplication involves transient dedifferentiation. Second, genetic and pharmacologic tools will be used to dissect the signaling pathways controlling the entry of beta cells to the cell division cycle, with emphasis on the roles of glucose and insulin, the key physiological input and output of beta cells. The expected outcome of these studies is a detailed molecular understanding of the homeostatic maintenance of beta cell mass, describing how beta cell function is linked to beta cell number in vivo. This may suggest new targets and concepts for pharmacologic intervention, towards the development of regenerative therapy strategies in diabetes. More generally, the experiments will shed light on one of the greatest mysteries of developmental biology, namely how organs achieve and maintain their correct size.

Max ERC Funding

1 445 000 €

Duration

Start date: 2010-09-01, End date: 2015-08-31

Project acronymCaNANObinoids

ProjectFrom Peripheralized to Cell- and Organelle-Targeted Medicine: The 3rd Generation of Cannabinoid-1 Receptor Antagonists for the Treatment of Chronic Kidney Disease

Researcher (PI)Yossef Tam

Host Institution (HI)THE HEBREW UNIVERSITY OF JERUSALEM

Call DetailsStarting Grant (StG), LS4, ERC-2015-STG

SummaryClinical experience with globally-acting cannabinoid-1 receptor (CB1R) antagonists revealed the benefits of blocking CB1Rs for the treatment of obesity and diabetes. However, their use is hampered by increased CNS-mediated side effects. Recently, I have demonstrated that peripherally-restricted CB1R antagonists have the potential to treat the metabolic syndrome without eliciting these adverse effects. While these results are promising and are currently being developed into the clinic, our ability to rationally design CB1R blockers that would target a diseased organ is limited.
The current proposal aims to develop and test cell- and organelle-specific CB1R antagonists. To establish this paradigm, I will focus our interest on the kidney, since chronic kidney disease (CKD) is the leading cause of increased morbidity and mortality of patients with diabetes. Our first goal will be to characterize the obligatory role of the renal proximal tubular CB1R in the pathogenesis of diabetic renal complications. Next, we will attempt to link renal proximal CB1R with diabetic mitochondrial dysfunction. Finally, we will develop proximal tubular (cell-specific) and mitochondrial (organelle-specific) CB1R blockers and test their effectiveness in treating CKD. To that end, we will encapsulate CB1R blockers into biocompatible polymeric nanoparticles that will serve as targeted drug delivery systems, via their conjugation to targeting ligands.
The implications of this work are far reaching as they will (i) point to renal proximal tubule CB1R as a novel target for CKD; (ii) identify mitochondrial CB1R as a new player in the regulation of proximal tubular cell function, and (iii) eventually become the drug-of-choice in treating diabetic CKD and its comorbidities. Moreover, this work will lead to the development of a novel organ-specific drug delivery system for CB1R blockers, which could be then exploited in other tissues affected by obesity, diabetes and the metabolic syndrome.

Clinical experience with globally-acting cannabinoid-1 receptor (CB1R) antagonists revealed the benefits of blocking CB1Rs for the treatment of obesity and diabetes. However, their use is hampered by increased CNS-mediated side effects. Recently, I have demonstrated that peripherally-restricted CB1R antagonists have the potential to treat the metabolic syndrome without eliciting these adverse effects. While these results are promising and are currently being developed into the clinic, our ability to rationally design CB1R blockers that would target a diseased organ is limited.
The current proposal aims to develop and test cell- and organelle-specific CB1R antagonists. To establish this paradigm, I will focus our interest on the kidney, since chronic kidney disease (CKD) is the leading cause of increased morbidity and mortality of patients with diabetes. Our first goal will be to characterize the obligatory role of the renal proximal tubular CB1R in the pathogenesis of diabetic renal complications. Next, we will attempt to link renal proximal CB1R with diabetic mitochondrial dysfunction. Finally, we will develop proximal tubular (cell-specific) and mitochondrial (organelle-specific) CB1R blockers and test their effectiveness in treating CKD. To that end, we will encapsulate CB1R blockers into biocompatible polymeric nanoparticles that will serve as targeted drug delivery systems, via their conjugation to targeting ligands.
The implications of this work are far reaching as they will (i) point to renal proximal tubule CB1R as a novel target for CKD; (ii) identify mitochondrial CB1R as a new player in the regulation of proximal tubular cell function, and (iii) eventually become the drug-of-choice in treating diabetic CKD and its comorbidities. Moreover, this work will lead to the development of a novel organ-specific drug delivery system for CB1R blockers, which could be then exploited in other tissues affected by obesity, diabetes and the metabolic syndrome.

SummaryThe study of several genetic disorders is hampered by the lack of suitable in vitro human models. We hypothesize that the generation of patient-specific induced pluripotent stem cells (iPSCs) will allow the development of disease-specific in vitro models; yielding new pathophysiologic insights into several genetic disorders and offering a unique platform to test novel therapeutic strategies. In the current proposal we plan utilize this novel approach to establish human iPSC (hiPSC) lines for the study of a variety of inherited cardiac disorders. The specific disease states that will be studied were chosen to reflect abnormalities in a wide-array of different cardiomyocyte cellular processes.
These include mutations leading to:
(1) abnormal ion channel function (“channelopathies”), such as the long QT and Brugada syndromes;
(2) abnormal intracellular storage of unnecessary material, such as in the glycogen storage disease type IIb (Pompe’s disease); and
(3) abnormalities in cell-to-cell contacts, such as in the case of arrhythmogenic right ventricular cardiomyopathy-dysplasia (ARVC-D). The different hiPSC lines generated will be coaxed to differentiate into the cardiac lineage. Detailed molecular, structural, functional, and pharmacological studies will then be performed to characterize the phenotypic properties of the generated hiPSC-derived cardiomyocytes, with specific emphasis on their molecular, ultrastructural, electrophysiological, and Ca2+ handling properties.
These studies should provide new insights into the pathophysiological mechanisms underlying the different familial arrhythmogenic and cardiomyopathy disorders studied, may allow optimization of patient-specific therapies (personalized medicine), and may facilitate the development of novel therapeutic strategies.
Moreover, the concepts and methodological knowhow developed in the current project could be extended, in the future, to derive human disease-specific cell culture models for a plurality of genetic disorders; enabling translational research ranging from investigation of the most fundamental cellular mechanisms involved in human tissue formation and physiology through disease investigation and the development and testing of novel therapies that could potentially find their way to the bedside

The study of several genetic disorders is hampered by the lack of suitable in vitro human models. We hypothesize that the generation of patient-specific induced pluripotent stem cells (iPSCs) will allow the development of disease-specific in vitro models; yielding new pathophysiologic insights into several genetic disorders and offering a unique platform to test novel therapeutic strategies. In the current proposal we plan utilize this novel approach to establish human iPSC (hiPSC) lines for the study of a variety of inherited cardiac disorders. The specific disease states that will be studied were chosen to reflect abnormalities in a wide-array of different cardiomyocyte cellular processes.
These include mutations leading to:
(1) abnormal ion channel function (“channelopathies”), such as the long QT and Brugada syndromes;
(2) abnormal intracellular storage of unnecessary material, such as in the glycogen storage disease type IIb (Pompe’s disease); and
(3) abnormalities in cell-to-cell contacts, such as in the case of arrhythmogenic right ventricular cardiomyopathy-dysplasia (ARVC-D). The different hiPSC lines generated will be coaxed to differentiate into the cardiac lineage. Detailed molecular, structural, functional, and pharmacological studies will then be performed to characterize the phenotypic properties of the generated hiPSC-derived cardiomyocytes, with specific emphasis on their molecular, ultrastructural, electrophysiological, and Ca2+ handling properties.
These studies should provide new insights into the pathophysiological mechanisms underlying the different familial arrhythmogenic and cardiomyopathy disorders studied, may allow optimization of patient-specific therapies (personalized medicine), and may facilitate the development of novel therapeutic strategies.
Moreover, the concepts and methodological knowhow developed in the current project could be extended, in the future, to derive human disease-specific cell culture models for a plurality of genetic disorders; enabling translational research ranging from investigation of the most fundamental cellular mechanisms involved in human tissue formation and physiology through disease investigation and the development and testing of novel therapies that could potentially find their way to the bedside

Max ERC Funding

1 500 000 €

Duration

Start date: 2011-03-01, End date: 2016-02-29

Project acronymCFS modelling

ProjectChromosomal Common Fragile Sites: Unravelling their biological functions and the basis of their instability

Researcher (PI)Andres Joaquin Lopez-Contreras

Host Institution (HI)KOBENHAVNS UNIVERSITET

Call DetailsStarting Grant (StG), LS4, ERC-2015-STG

SummaryCancer and other diseases are driven by genomic alterations initiated by DNA breaks. Within our genomes, some regions are particularly prone to breakage, and these are known as common fragile sites (CFSs). CFSs are present in every person and are frequently sites of oncogenic chromosomal rearrangements. Intriguingly, despite their fragility, many CFSs are well conserved through evolution, suggesting that these regions have important physiological functions that remain elusive. My previous background in genome editing, proteomics and replication-born DNA damage has given me the tools to propose an ambitious and comprehensive plan that tackles fundamental questions on the biology of CFSs. First, we will perform a systematic analysis of the function of CFSs. Most of the CFSs contain very large genes, which has made technically difficult to dissect whether the CFS role is due to the locus itself or to the encoded gene product. However, the emergence of the CRISPR/Cas9 technology now enables the study of CFSs on a more systematic basis. We will pioneer the engineering of mammalian models harbouring large deletions at CFS loci to investigate their physiological functions at the cellular and organism levels. For those CFSs that contain genes, the cDNAs will be re-introduced at a distal locus. Using this strategy, we will be able to achieve the first comprehensive characterization of CFS roles. Second, we will develop novel targeted approaches to interrogate the chromatin-bound proteome of CFSs and its dynamics during DNA replication. Finally, and given that CFS fragility is influenced both by cell cycle checkpoints and dNTP availability, we will use mouse models to study the impact of ATR/CHK1 pathway and dNTP levels on CFS instability and cancer. Taken together, I propose an ambitious, yet feasible, project to functionally annotate and characterise these poorly understood regions of the human genome, with important potential implications for improving human health.

Cancer and other diseases are driven by genomic alterations initiated by DNA breaks. Within our genomes, some regions are particularly prone to breakage, and these are known as common fragile sites (CFSs). CFSs are present in every person and are frequently sites of oncogenic chromosomal rearrangements. Intriguingly, despite their fragility, many CFSs are well conserved through evolution, suggesting that these regions have important physiological functions that remain elusive. My previous background in genome editing, proteomics and replication-born DNA damage has given me the tools to propose an ambitious and comprehensive plan that tackles fundamental questions on the biology of CFSs. First, we will perform a systematic analysis of the function of CFSs. Most of the CFSs contain very large genes, which has made technically difficult to dissect whether the CFS role is due to the locus itself or to the encoded gene product. However, the emergence of the CRISPR/Cas9 technology now enables the study of CFSs on a more systematic basis. We will pioneer the engineering of mammalian models harbouring large deletions at CFS loci to investigate their physiological functions at the cellular and organism levels. For those CFSs that contain genes, the cDNAs will be re-introduced at a distal locus. Using this strategy, we will be able to achieve the first comprehensive characterization of CFS roles. Second, we will develop novel targeted approaches to interrogate the chromatin-bound proteome of CFSs and its dynamics during DNA replication. Finally, and given that CFS fragility is influenced both by cell cycle checkpoints and dNTP availability, we will use mouse models to study the impact of ATR/CHK1 pathway and dNTP levels on CFS instability and cancer. Taken together, I propose an ambitious, yet feasible, project to functionally annotate and characterise these poorly understood regions of the human genome, with important potential implications for improving human health.

SummaryTetralogy of Fallot (TOF) is the most common congenital heart disease (CHD) occurring 1 in 3000 births. Genetic studies have identified numerous genes that are responsible for inherited and sporadic forms of TOF, most of which encode key molecules that are part of regulatory networks controlling heart development. The identification of two populations of cardiac precursors, one exclusively forming the left ventricle and the second the outflow tract, the right ventricle and the atria, has suggested a new approach to interpret CHDs, in particular in TOF, not as a defect in a specific gene, but rather as a defect in the formation, expansion, and differentiation of defined subsets of embryonic cardiac precursors. The LIM-homeodomain transcription factor ISL1 marks the second population of cardiac progenitors, but little is known about its downstream targets, and how causative genes of CHDs affect cell-fate decisions in the ISL1 lineage. The main goals of this research program are: (1) to decipher the functional role of Isl1 downstream targets identified by a genome-wide ChIP-Seq approach; (2) to generate induced pluripotent stem (iPS) cells from controls and patients affected by severe forms of TOF characterized by defects in heart compartments known to derive from ISL1 cardiac progenitors; (3) to direct these iPS cells to ISL1+ cardiovascular precursors and identify cell-surface makers enabling their antibody-based purification; and (4) to use TOF-iPS-derived ISL1+ progenitors as an unique in vitro model system for deciphering molecular mechanisms that govern the fates and differentiation of this progenitor lineage and determine the pathological phenotype seen in TOF. This work will shed light on the molecular mechanisms of ISL1+ cardiac progenitor lineage specification and will give important new insights into the mechanisms of how alterations in transcriptional and epigenetic programs translate to a distinct structural defect during cardiogenesis.

Tetralogy of Fallot (TOF) is the most common congenital heart disease (CHD) occurring 1 in 3000 births. Genetic studies have identified numerous genes that are responsible for inherited and sporadic forms of TOF, most of which encode key molecules that are part of regulatory networks controlling heart development. The identification of two populations of cardiac precursors, one exclusively forming the left ventricle and the second the outflow tract, the right ventricle and the atria, has suggested a new approach to interpret CHDs, in particular in TOF, not as a defect in a specific gene, but rather as a defect in the formation, expansion, and differentiation of defined subsets of embryonic cardiac precursors. The LIM-homeodomain transcription factor ISL1 marks the second population of cardiac progenitors, but little is known about its downstream targets, and how causative genes of CHDs affect cell-fate decisions in the ISL1 lineage. The main goals of this research program are: (1) to decipher the functional role of Isl1 downstream targets identified by a genome-wide ChIP-Seq approach; (2) to generate induced pluripotent stem (iPS) cells from controls and patients affected by severe forms of TOF characterized by defects in heart compartments known to derive from ISL1 cardiac progenitors; (3) to direct these iPS cells to ISL1+ cardiovascular precursors and identify cell-surface makers enabling their antibody-based purification; and (4) to use TOF-iPS-derived ISL1+ progenitors as an unique in vitro model system for deciphering molecular mechanisms that govern the fates and differentiation of this progenitor lineage and determine the pathological phenotype seen in TOF. This work will shed light on the molecular mechanisms of ISL1+ cardiac progenitor lineage specification and will give important new insights into the mechanisms of how alterations in transcriptional and epigenetic programs translate to a distinct structural defect during cardiogenesis.

Max ERC Funding

1 499 996 €

Duration

Start date: 2011-03-01, End date: 2017-02-28

Project acronymCOMPLEXI&AGING

ProjectModulation of mitochondrial complex I as a strategy to increase lifespan and prevent age-related diseases

Researcher (PI)Alberto Sanz Montero

Host Institution (HI)UNIVERSITY OF NEWCASTLE UPON TYNE

Call DetailsStarting Grant (StG), LS4, ERC-2010-StG_20091118

SummaryNowadays, ageing is one of the main problems in Western society. The increase in the percentage of elderly people serves to strain the Social Security to the point of bankruptcy. The only way to alleviate the suffering caused by age-related degenerative disease is to fully understand the underlying forces which drive ageing and design strategies to delay it. Mitochondria are considered as central modulators of longevity in different species. It has been proposed that free radicals cause the accumulation of oxidative damage and as a result ageing. In accordance with this, production of Reactive Oxygen Species (ROS) by complex I negatively correlates with longevity. However, the overexpression of antioxidants or the reduction of ROS levels does not increase lifespan. These contradictory data can only be reconciled if complex I is modulating longevity through a ROS independent mechanism. We have expressed the alternative internal NADH dehydrogenase 1 (NDI1) from Saccharomyces cerevisiae in Drosophila melanogaster. The expression of NDI1 does not change the level of ROS but increases both the ratio of NAD+/NADH and Drosophila longevity. The main objective of this proposal is to study the mechanisms by which complex I regulates longevity. My general hypothesis is that complex I regulates longevity through a ROS independent mechanism. I propose that complex I controls the cellular levels of NAD+/NADH, keeping their levels at an equilibrium that favours the optimal functioning of the cell. When the ratio is moved towards NADH ageing is promoted, whereas when it is moved towards NAD+ pro-survival pathways are activated. I proposed two specific mechanisms downstream of complex I that promote cellular longevity or senescence: 1) activation of sirtuins, which would increase genome stability and 2) reduction of methylglyoxal generation, which would decrease the accumulation of cellular garbarge .

Nowadays, ageing is one of the main problems in Western society. The increase in the percentage of elderly people serves to strain the Social Security to the point of bankruptcy. The only way to alleviate the suffering caused by age-related degenerative disease is to fully understand the underlying forces which drive ageing and design strategies to delay it. Mitochondria are considered as central modulators of longevity in different species. It has been proposed that free radicals cause the accumulation of oxidative damage and as a result ageing. In accordance with this, production of Reactive Oxygen Species (ROS) by complex I negatively correlates with longevity. However, the overexpression of antioxidants or the reduction of ROS levels does not increase lifespan. These contradictory data can only be reconciled if complex I is modulating longevity through a ROS independent mechanism. We have expressed the alternative internal NADH dehydrogenase 1 (NDI1) from Saccharomyces cerevisiae in Drosophila melanogaster. The expression of NDI1 does not change the level of ROS but increases both the ratio of NAD+/NADH and Drosophila longevity. The main objective of this proposal is to study the mechanisms by which complex I regulates longevity. My general hypothesis is that complex I regulates longevity through a ROS independent mechanism. I propose that complex I controls the cellular levels of NAD+/NADH, keeping their levels at an equilibrium that favours the optimal functioning of the cell. When the ratio is moved towards NADH ageing is promoted, whereas when it is moved towards NAD+ pro-survival pathways are activated. I proposed two specific mechanisms downstream of complex I that promote cellular longevity or senescence: 1) activation of sirtuins, which would increase genome stability and 2) reduction of methylglyoxal generation, which would decrease the accumulation of cellular garbarge .

SummaryChronic kidney disease (CKD) is a growing public health problem with a massively increased cardiovascular mortality. Patients with advanced CKD mostly die from sudden cardiac death and recurrent heart failure due to premature cardiac aging with hypertrophy, fibrosis, and capillary rarefaction. I have recently identified the long sought key cardiac myofibroblast progenitor population, an emerging breakthrough that carries the potential to develop novel targeted therapeutics. Genetic ablation of these Gli1+ perivascular progenitors ameliorates fibrosis, cardiac hypertrophy and rescues left-ventricular function. I propose that Gli1+ cells are critically involved in all major pathophysiologic changes in cardiac aging and uremic cardiomyopathy including fibrosis, hypertrophy and capillary rarefaction. I will perform state of the art genetic fate tracing, ablation and in vivo CRISPR/Cas9 genome editing experiments to untangle their complex mechanism of activation and communication with endothelial cells and cardiomyocytes promoting fibrosis, capillary rarefaction, cardiac hypertrophy and heart failure. To identify novel druggable targets I will utilize new mouse models that allow comparative transcript and proteasome profiling assays of these critical myofibroblast precusors in homeostasis, aging and premature aging in CKD. Novel assays with immortalized cardiac Gli1+ cells will allow high throughput screens to identify uremia associated factors of cell activation and inhibitory compounds to facilitate the development of novel therapeutics.
This ambitious interdisciplinary project requires the expertise of chemists, physiologists, biomedical researchers and physician scientists to develop novel targeted therapies in cardiac remodeling during aging and CKD. The passion that drives this project results from a simple emerging hypothesis: It is possible to treat heart failure and sudden cardiac death in aging and CKD by targeting perivascular myofibroblast progenitors.

Chronic kidney disease (CKD) is a growing public health problem with a massively increased cardiovascular mortality. Patients with advanced CKD mostly die from sudden cardiac death and recurrent heart failure due to premature cardiac aging with hypertrophy, fibrosis, and capillary rarefaction. I have recently identified the long sought key cardiac myofibroblast progenitor population, an emerging breakthrough that carries the potential to develop novel targeted therapeutics. Genetic ablation of these Gli1+ perivascular progenitors ameliorates fibrosis, cardiac hypertrophy and rescues left-ventricular function. I propose that Gli1+ cells are critically involved in all major pathophysiologic changes in cardiac aging and uremic cardiomyopathy including fibrosis, hypertrophy and capillary rarefaction. I will perform state of the art genetic fate tracing, ablation and in vivo CRISPR/Cas9 genome editing experiments to untangle their complex mechanism of activation and communication with endothelial cells and cardiomyocytes promoting fibrosis, capillary rarefaction, cardiac hypertrophy and heart failure. To identify novel druggable targets I will utilize new mouse models that allow comparative transcript and proteasome profiling assays of these critical myofibroblast precusors in homeostasis, aging and premature aging in CKD. Novel assays with immortalized cardiac Gli1+ cells will allow high throughput screens to identify uremia associated factors of cell activation and inhibitory compounds to facilitate the development of novel therapeutics.
This ambitious interdisciplinary project requires the expertise of chemists, physiologists, biomedical researchers and physician scientists to develop novel targeted therapies in cardiac remodeling during aging and CKD. The passion that drives this project results from a simple emerging hypothesis: It is possible to treat heart failure and sudden cardiac death in aging and CKD by targeting perivascular myofibroblast progenitors.

Max ERC Funding

1 497 888 €

Duration

Start date: 2016-05-01, End date: 2021-04-30

Project acronymEVI1inCancer

ProjectOvercoming the epigenetic and therapeutic barrier of EVI1-overexpressing cancers

Researcher (PI)Stefan Gröschel

Host Institution (HI)DEUTSCHES KREBSFORSCHUNGSZENTRUM HEIDELBERG

Call DetailsStarting Grant (StG), LS4, ERC-2015-STG

SummaryDeregulation of the EVI1 oncogene is a key transforming event in the development of many malignancies, most prominently very high-risk acute myeloid leukemia (AML), ovarian, colon, breast, non-small cell lung cancer, and soft-tissue sarcoma. For decades, both EVI1 function and the mechanism underlying its deregulation have been poorly understood. The consequent lack of a targeted therapy against EVI1 establishes a pressing medical need. In a recent study investigating a distinct category of EVI1-driven AML with inv(3) or t(3;3), we characterized the regulatory domain of EVI1 and identified a master regulatory element of the stemness factor GATA2 to be rearranged to EVI1, thereby deregulating both genes. Applying functional genomics and genome-editing, we found that the rearranged enhancer element adopted novel features, such as superloading of the epigenetic reader and chromatin regulator BRD4, allowing its inhibition with BET/bromodomain inhibitors with relative EVI1 specificity. Interference with EVI1-regulatory mechanisms thus has potential therapeutic value in EVI1-transformed tumors. To pave the way for epigenetic targeting of other EVI1-expressing malignancies, we aim to identify genomic enhancer sequences and protein components of the EVI1 regulatory domain by systematic epigenetic and proteomic profiling. Specifically, we seek to achieve the following experimental goals: (1) Identification of the mechanism underlying EVI1 deregulation in non-3q-rearranged AML and solid tumors; (2) Addressing the role of breakpoint-associated transpos-able retroelements; (3) Characterization of the transcription factor complex regulating EVI1; (4) Identification of epigenetic resistance mechanisms in EVI1+ AML by using an in vivo model and a genome-editing approach. The proposed experiments will provide insight into the epigenetic landscape of EVI1+ malignancies and help reveal new targets and genetic interactions amenable to future therapies in these high-risk malignancies.

Deregulation of the EVI1 oncogene is a key transforming event in the development of many malignancies, most prominently very high-risk acute myeloid leukemia (AML), ovarian, colon, breast, non-small cell lung cancer, and soft-tissue sarcoma. For decades, both EVI1 function and the mechanism underlying its deregulation have been poorly understood. The consequent lack of a targeted therapy against EVI1 establishes a pressing medical need. In a recent study investigating a distinct category of EVI1-driven AML with inv(3) or t(3;3), we characterized the regulatory domain of EVI1 and identified a master regulatory element of the stemness factor GATA2 to be rearranged to EVI1, thereby deregulating both genes. Applying functional genomics and genome-editing, we found that the rearranged enhancer element adopted novel features, such as superloading of the epigenetic reader and chromatin regulator BRD4, allowing its inhibition with BET/bromodomain inhibitors with relative EVI1 specificity. Interference with EVI1-regulatory mechanisms thus has potential therapeutic value in EVI1-transformed tumors. To pave the way for epigenetic targeting of other EVI1-expressing malignancies, we aim to identify genomic enhancer sequences and protein components of the EVI1 regulatory domain by systematic epigenetic and proteomic profiling. Specifically, we seek to achieve the following experimental goals: (1) Identification of the mechanism underlying EVI1 deregulation in non-3q-rearranged AML and solid tumors; (2) Addressing the role of breakpoint-associated transpos-able retroelements; (3) Characterization of the transcription factor complex regulating EVI1; (4) Identification of epigenetic resistance mechanisms in EVI1+ AML by using an in vivo model and a genome-editing approach. The proposed experiments will provide insight into the epigenetic landscape of EVI1+ malignancies and help reveal new targets and genetic interactions amenable to future therapies in these high-risk malignancies.

SummaryHIF transcription factors, central mediators of cellular adaptation to critically low oxygen levels (=hypoxia), have been largely studied for their crucial role during cancer development. Our recent findings have unveiled two new major roles of HIF. First, in innate immunity and infection, we demonstrated the key contributions of HIFs in regulating important immune effectors molecules. Second, we highlighted the role of HIFs in iron metabolism as critical regulators of iron absorption in the intestine and in systemic iron homeostasis by regulating the liver synthesis of the iron regulatory hormone, hepcidin.
These results open new research areas and our research program, based on unique mouse models of conditional HIFs and hepcidin knockout, will be developed around three main axes.
1) define the physiological roles of HIF and hepcidin in different key organs involved in maintaining body iron homeostasis. A detailed understanding of the regulation of iron-related proteins is a prerequisite in the development of therapeutics for iron diseases, which pose a major problem worldwide.
2) study the role of hepcidin during bacterial infection and tumorigenesis, two pathological conditions where iron is critically required for the proliferation of the pathogens and for cancer cells to feed their high metabolic activity.
3) determine the contribution of HIFs in the initiation of tumor development in response to infection. Indeed, our findings that HIF is stabilized by bacteria, even under normal levels of oxygen, and is an essential component of the inflammation response, let us to speculate that HIFs may be a missing link between infection and cancer by triggering a high chronic inflammatory response. For that, we will use the model of the gastric cancer, whose the initiating event is an infection by Helicobacter Pylori.

HIF transcription factors, central mediators of cellular adaptation to critically low oxygen levels (=hypoxia), have been largely studied for their crucial role during cancer development. Our recent findings have unveiled two new major roles of HIF. First, in innate immunity and infection, we demonstrated the key contributions of HIFs in regulating important immune effectors molecules. Second, we highlighted the role of HIFs in iron metabolism as critical regulators of iron absorption in the intestine and in systemic iron homeostasis by regulating the liver synthesis of the iron regulatory hormone, hepcidin.
These results open new research areas and our research program, based on unique mouse models of conditional HIFs and hepcidin knockout, will be developed around three main axes.
1) define the physiological roles of HIF and hepcidin in different key organs involved in maintaining body iron homeostasis. A detailed understanding of the regulation of iron-related proteins is a prerequisite in the development of therapeutics for iron diseases, which pose a major problem worldwide.
2) study the role of hepcidin during bacterial infection and tumorigenesis, two pathological conditions where iron is critically required for the proliferation of the pathogens and for cancer cells to feed their high metabolic activity.
3) determine the contribution of HIFs in the initiation of tumor development in response to infection. Indeed, our findings that HIF is stabilized by bacteria, even under normal levels of oxygen, and is an essential component of the inflammation response, let us to speculate that HIFs may be a missing link between infection and cancer by triggering a high chronic inflammatory response. For that, we will use the model of the gastric cancer, whose the initiating event is an infection by Helicobacter Pylori.

SummaryBreast cancer is the most common cancer in women, resulting in as many as 500000 deaths per year worldwide. Patients with breast cancer die unequivocally because of the development of incurable distant metastases and not because of symptoms related to the primary site. Understanding the complex, yet fundamental mechanisms driving breast cancer metastasis is critical to develop therapies tailored to this disease.
The current understanding of how metastasis occurs is derived primarily from mouse models and largely dominated by the notion that single migratory cancer cells within the primary tumor can actively disseminate to distant sites and develop as metastatic deposits. Unexpectedly, our very recent study on patient blood samples has shown that cancer cell groupings, held together through strong cell-cell junctions, can break off the primary tumor and form a metastatic lesion up to 50 times more efficiently than single migratory cancer cells (Aceto et al, Cell, 2014). These findings lead to new open questions, yet highlight a previously unappreciated and targetable mechanism of cancer dissemination.
Our preliminary data suggest that, among all types of cell-cell junctions, desmosomes and tight junctions are involved in this process, and therefore represent unprecedented options for developing a metastasis-tailored therapy for breast cancer.
The two predominant goals of this proposal are: first, to define the role of specific desmosome (DSG2) and tight junction (CLDN3 and TJP2) components in the development of metastasis. Second, to address their involvement in cellular signaling and response to therapy. These studies will not only use our first-of-a-kind in vivo models developed from patients with breast cancer metastases, but also cross the boundaries between basic science and clinical applications.
Our research has the long-term ambition to lead to a novel class of therapeutic agents tailored to block cell-cell junctions and prevent metastatic spread of cancer.

Breast cancer is the most common cancer in women, resulting in as many as 500000 deaths per year worldwide. Patients with breast cancer die unequivocally because of the development of incurable distant metastases and not because of symptoms related to the primary site. Understanding the complex, yet fundamental mechanisms driving breast cancer metastasis is critical to develop therapies tailored to this disease.
The current understanding of how metastasis occurs is derived primarily from mouse models and largely dominated by the notion that single migratory cancer cells within the primary tumor can actively disseminate to distant sites and develop as metastatic deposits. Unexpectedly, our very recent study on patient blood samples has shown that cancer cell groupings, held together through strong cell-cell junctions, can break off the primary tumor and form a metastatic lesion up to 50 times more efficiently than single migratory cancer cells (Aceto et al, Cell, 2014). These findings lead to new open questions, yet highlight a previously unappreciated and targetable mechanism of cancer dissemination.
Our preliminary data suggest that, among all types of cell-cell junctions, desmosomes and tight junctions are involved in this process, and therefore represent unprecedented options for developing a metastasis-tailored therapy for breast cancer.
The two predominant goals of this proposal are: first, to define the role of specific desmosome (DSG2) and tight junction (CLDN3 and TJP2) components in the development of metastasis. Second, to address their involvement in cellular signaling and response to therapy. These studies will not only use our first-of-a-kind in vivo models developed from patients with breast cancer metastases, but also cross the boundaries between basic science and clinical applications.
Our research has the long-term ambition to lead to a novel class of therapeutic agents tailored to block cell-cell junctions and prevent metastatic spread of cancer.

Max ERC Funding

1 744 921 €

Duration

Start date: 2016-03-01, End date: 2021-02-28

Project acronymHUFATREG

ProjectAdipose tissue mass regulation in lean and obese individuals

Researcher (PI)Kirsty Lee Spalding

Host Institution (HI)KAROLINSKA INSTITUTET

Call DetailsStarting Grant (StG), LS4, ERC-2010-StG_20091118

SummaryOwing to the increase in obesity, life expectancy may start to decrease in developed countries for the first time in recent history. In humans the generation of fat cells (adipocytes) is a major factor behind the growth of adipose tissue during childhood. The factors determining the fat mass in adults, however, are not fully understood. Increased fat storage in fully differentiated adipocytes, resulting in enlarged fat cells, is well documented and thought to be the most important mechanism whereby fat depots increase in adults. Very little is known about the maintenance of fat cells (adipocytes) in humans, how different fat depots are maintained and how (or if) this is altered in obesity. Recently I developed a method that is based on the incorporation of 14C from nuclear bomb tests into genomic DNA, which allows for the analysis of cell and tissue turnover in humans. Using this novel methodology we now have a strategy for studying cell turnover in humans. One tissue of great interest and significant clinical relevance is adipose tissue. Excess adipose tissue, resulting in obesity, is currently one of the most serious threats to human health on a global level. The current proposal aims to determine the dynamics of human adipose tissue maintenance and investigate any differences in regulation of the fat mass in lean and obese individuals. Understanding the dynamics of adipocyte turnover may shed new light on potential treatments for obesity.

Owing to the increase in obesity, life expectancy may start to decrease in developed countries for the first time in recent history. In humans the generation of fat cells (adipocytes) is a major factor behind the growth of adipose tissue during childhood. The factors determining the fat mass in adults, however, are not fully understood. Increased fat storage in fully differentiated adipocytes, resulting in enlarged fat cells, is well documented and thought to be the most important mechanism whereby fat depots increase in adults. Very little is known about the maintenance of fat cells (adipocytes) in humans, how different fat depots are maintained and how (or if) this is altered in obesity. Recently I developed a method that is based on the incorporation of 14C from nuclear bomb tests into genomic DNA, which allows for the analysis of cell and tissue turnover in humans. Using this novel methodology we now have a strategy for studying cell turnover in humans. One tissue of great interest and significant clinical relevance is adipose tissue. Excess adipose tissue, resulting in obesity, is currently one of the most serious threats to human health on a global level. The current proposal aims to determine the dynamics of human adipose tissue maintenance and investigate any differences in regulation of the fat mass in lean and obese individuals. Understanding the dynamics of adipocyte turnover may shed new light on potential treatments for obesity.

Max ERC Funding

1 500 000 €

Duration

Start date: 2011-04-01, End date: 2017-03-31

Project acronymHYPOXICMICRORNAS

ProjectDeciphering the microRNA response to hypoxia

Researcher (PI)Roger David John Pocock

Host Institution (HI)KOBENHAVNS UNIVERSITET

Call DetailsStarting Grant (StG), LS4, ERC-2010-StG_20091118

SummaryMaintaining oxygen homeostasis is an essential requirement for all metazoa. Oxygen is required for efficient generation of energy, however, as oxygen levels decrease (hypoxia), cells mount a variety of adaptive responses. Each cell in the body can sense and respond to hypoxia, yet the molecular mechanisms that regulate these responses are only beginning to be delineated. Hypoxia plays crucial roles in the pathophysiology of cancer, neurological dysfunction, myocardial infarction and lung disease. Therefore, the goal of the proposed research is to better understand how cells sense and adapt to hypoxia. To this end, I am using the powerful genetic model of Caenorhabditis elegans to identify novel molecular mechanisms required for oxygen homeostatic responses.
A critical regulator of hypoxic responses in all cell types is the conserved hypoxia-inducible factor (HIF-1). In response to a hypoxic insult, HIF-1 transcriptionally regulates a wide variety of target genes to facilitate adaptation. Recent studies indicate that in addition to the canonical HIF-1 pathway, microRNAs (miRNAs) play important roles in hypoxic response mechanisms. miRNAs are regulatory molecules that predominantly repress protein production of their target genes, however, their roles in hypoxic adaptation are poorly understood. I recently found that specific phylogenetically conserved miRNAs are regulated by hypoxia in C. elegans; and that the function of these miRNAs is required for survival of animals in low oxygen conditions. This is truly an emerging field of science and I expect to make groundbreaking discoveries in the regulation of hypoxic and metabolic responses by miRNAs, which will improve our understanding of many disease processes.
The proposed research will 1) analyze the functional roles of specific miRNAs in hypoxic responses and 2) utilize immunoprecipitation, bioinformatics and genetic screening combined with state-of-the-art deep sequencing technology to identify novel miRNA targets required for adaptation to hypoxia.

Maintaining oxygen homeostasis is an essential requirement for all metazoa. Oxygen is required for efficient generation of energy, however, as oxygen levels decrease (hypoxia), cells mount a variety of adaptive responses. Each cell in the body can sense and respond to hypoxia, yet the molecular mechanisms that regulate these responses are only beginning to be delineated. Hypoxia plays crucial roles in the pathophysiology of cancer, neurological dysfunction, myocardial infarction and lung disease. Therefore, the goal of the proposed research is to better understand how cells sense and adapt to hypoxia. To this end, I am using the powerful genetic model of Caenorhabditis elegans to identify novel molecular mechanisms required for oxygen homeostatic responses.
A critical regulator of hypoxic responses in all cell types is the conserved hypoxia-inducible factor (HIF-1). In response to a hypoxic insult, HIF-1 transcriptionally regulates a wide variety of target genes to facilitate adaptation. Recent studies indicate that in addition to the canonical HIF-1 pathway, microRNAs (miRNAs) play important roles in hypoxic response mechanisms. miRNAs are regulatory molecules that predominantly repress protein production of their target genes, however, their roles in hypoxic adaptation are poorly understood. I recently found that specific phylogenetically conserved miRNAs are regulated by hypoxia in C. elegans; and that the function of these miRNAs is required for survival of animals in low oxygen conditions. This is truly an emerging field of science and I expect to make groundbreaking discoveries in the regulation of hypoxic and metabolic responses by miRNAs, which will improve our understanding of many disease processes.
The proposed research will 1) analyze the functional roles of specific miRNAs in hypoxic responses and 2) utilize immunoprecipitation, bioinformatics and genetic screening combined with state-of-the-art deep sequencing technology to identify novel miRNA targets required for adaptation to hypoxia.

SummaryMany reports indicate the number of people with diabetes will exceed 350 million by the year 2030. Both type 1 and type 2 diabetes are characterized by the deterioration and impaired function of pancreatic b-cells. While transplantation is a promising strategy to replace lost tissue, several obstacles remain in the pathway to its clinical application. Whether b-cells are derived from patient samples or differentiated from embryonic stem cells, a major concern facing these strategies is how a recipient will respond to transplanted foreign tissue. Since the native environment for pancreatic islets is comprised of neural and vascular networks, successful integration may depend upon signals received from these neighboring cell types. Using a multidisciplinary approach, the principal investigator plans to elucidate molecular mechanisms underlying the interactions between pancreatic islet cells and their neighboring endothelial cells. Developing an understanding of how these interactions change during the pathogenesis of disease will provide insight into how islet growth and insulin release is dependent upon signals received from adjacent cell types. Emphasis will be placed on genetic mouse models to measure changes in gene expression in both isolated pancreatic b-cells and endothelial cells to identify genes that mediate the interaction between these cell types. In addition, it is of great interest to identify secreted factors that may constitute autocrine or paracrine signalling mechanisms that influence growth and function between these cell types. Furthermore, it will be determined whether current protocols for the differentiation of mouse stem cells into insulin producing cells are improved by restoring the expression of genes which facilitate communication to endothelial cells. This project aims to identify genes essential to the vascular context of pancreatic b-cells to improve transplantation protocols and facilitate the development of therapeutic strategies for diabetes.

Many reports indicate the number of people with diabetes will exceed 350 million by the year 2030. Both type 1 and type 2 diabetes are characterized by the deterioration and impaired function of pancreatic b-cells. While transplantation is a promising strategy to replace lost tissue, several obstacles remain in the pathway to its clinical application. Whether b-cells are derived from patient samples or differentiated from embryonic stem cells, a major concern facing these strategies is how a recipient will respond to transplanted foreign tissue. Since the native environment for pancreatic islets is comprised of neural and vascular networks, successful integration may depend upon signals received from these neighboring cell types. Using a multidisciplinary approach, the principal investigator plans to elucidate molecular mechanisms underlying the interactions between pancreatic islet cells and their neighboring endothelial cells. Developing an understanding of how these interactions change during the pathogenesis of disease will provide insight into how islet growth and insulin release is dependent upon signals received from adjacent cell types. Emphasis will be placed on genetic mouse models to measure changes in gene expression in both isolated pancreatic b-cells and endothelial cells to identify genes that mediate the interaction between these cell types. In addition, it is of great interest to identify secreted factors that may constitute autocrine or paracrine signalling mechanisms that influence growth and function between these cell types. Furthermore, it will be determined whether current protocols for the differentiation of mouse stem cells into insulin producing cells are improved by restoring the expression of genes which facilitate communication to endothelial cells. This project aims to identify genes essential to the vascular context of pancreatic b-cells to improve transplantation protocols and facilitate the development of therapeutic strategies for diabetes.

SummaryThis research grant has two major aspects. The first seeks to understand the molecular and cellular basis of the evolution of clear cell renal cell carcinoma(ccRCC), the most frequent form of kidney cancer. We will utilise an integrated approach based on mouse genetics, the use of primary kidney epithelial cell culture systems, genetic screening approaches using RNA interference libraries and analysis of the genetic and molecular changes that arise in human kidney tumours. The rationale behind these studies is that by better understanding the molecular causes of ccRCC it will be possible to identify new molecules or signaling pathways that could serve as appropriate therapeutic targets. The second aspect of this grant relates to the development of a flexible experimental platform that will allow the rapid and simultaneous up- and down-regulation of gene expression in the mouse kidney in a manner in which the affected cells are marked by a luminescent marker. This system will be based on the injection of modified lentiviral gene overexpression and gene knockdown vectors, allowing us to exploit recently-developed genome-wide cDNA libraries and RNA interference libraries. This experimental system should be equally applicable to other organ systems and will allow for the first time a systematic approach to the manipulation of gene expression in living mice, additionally bypassing the time limitations associated with conventional mouse genetic approaches. We aim to develop this system within the biological context of this grant and will combine it with live-animal imaging approaches to generate a series of mouse models of ccRCC. These will ultimately serve as invaluable tools for testing novel therapeutic approaches against this currently untreatable disease.

This research grant has two major aspects. The first seeks to understand the molecular and cellular basis of the evolution of clear cell renal cell carcinoma(ccRCC), the most frequent form of kidney cancer. We will utilise an integrated approach based on mouse genetics, the use of primary kidney epithelial cell culture systems, genetic screening approaches using RNA interference libraries and analysis of the genetic and molecular changes that arise in human kidney tumours. The rationale behind these studies is that by better understanding the molecular causes of ccRCC it will be possible to identify new molecules or signaling pathways that could serve as appropriate therapeutic targets. The second aspect of this grant relates to the development of a flexible experimental platform that will allow the rapid and simultaneous up- and down-regulation of gene expression in the mouse kidney in a manner in which the affected cells are marked by a luminescent marker. This system will be based on the injection of modified lentiviral gene overexpression and gene knockdown vectors, allowing us to exploit recently-developed genome-wide cDNA libraries and RNA interference libraries. This experimental system should be equally applicable to other organ systems and will allow for the first time a systematic approach to the manipulation of gene expression in living mice, additionally bypassing the time limitations associated with conventional mouse genetic approaches. We aim to develop this system within the biological context of this grant and will combine it with live-animal imaging approaches to generate a series of mouse models of ccRCC. These will ultimately serve as invaluable tools for testing novel therapeutic approaches against this currently untreatable disease.

SummaryMalignant pleural effusion (MPE) is a significant problem most commonly caused by adenocarcinomas. Although tumors involving the pleura vary in their ability to produce MPE, pathways critical for MPE formation are poorly defined. We have found that mouse tumors harboring mutant (”)KRas produce MPE in mice while tumors without ”KRas do not. LLC and MC38 lung and colon adenocarcinomas, potent inducers of MPE in syngeneic mice, harbor ”KRas that drives constitutive Ras and alternative nuclear factor (NF)-ºB signaling, inflammatory gene expression, and recruitment of specific myeloid cells to the pleural space. In contrast, mouse B16 melanoma and AE17 mesothelioma have wtKRas, lack constitutive Ras/alternative NF-º’ signaling, and are incapable of forming MPE. RNAi-mediated silencing of KRas in MC38 tumors abrogated MPE formation and Ras/alternative NF-º’ activation, while these phenomena were reconstituted in B16 tumors after KRas overexpression. We hypothesize that Ras-activating mutations drive the inflammatory phenotype of adenocarcinomas critical for MPE formation, which is characterized by Ras/alternative NF-ºB activation, inflammatory signalling to host vasculature/immune system, and recruitment of specific myeloid cells, and results in endothelial proliferation/leakiness. To test this hypothesis, we propose to: 1) define the relationship between Ras-activating mutations (RAM) and MPE formation; 2) identify tumor cell Ras-dependent signalling pathways and gene expression signature critical for MPE formation; 3) investigate the host response to tumor cells with RAM that results in MPE; and 4) target Ras and dependent signalling pathways as potential therapy for MPE. Studies will be performed using delivery of mouse/human tumors with/without RAM into the pleura of syngeneic/immunocompromized mice and are likely to yield new insights into the mechanisms of pleural tumor progression and to identify novel approaches to treatment of cancer patients with MPE.

Malignant pleural effusion (MPE) is a significant problem most commonly caused by adenocarcinomas. Although tumors involving the pleura vary in their ability to produce MPE, pathways critical for MPE formation are poorly defined. We have found that mouse tumors harboring mutant (”)KRas produce MPE in mice while tumors without ”KRas do not. LLC and MC38 lung and colon adenocarcinomas, potent inducers of MPE in syngeneic mice, harbor ”KRas that drives constitutive Ras and alternative nuclear factor (NF)-ºB signaling, inflammatory gene expression, and recruitment of specific myeloid cells to the pleural space. In contrast, mouse B16 melanoma and AE17 mesothelioma have wtKRas, lack constitutive Ras/alternative NF-º’ signaling, and are incapable of forming MPE. RNAi-mediated silencing of KRas in MC38 tumors abrogated MPE formation and Ras/alternative NF-º’ activation, while these phenomena were reconstituted in B16 tumors after KRas overexpression. We hypothesize that Ras-activating mutations drive the inflammatory phenotype of adenocarcinomas critical for MPE formation, which is characterized by Ras/alternative NF-ºB activation, inflammatory signalling to host vasculature/immune system, and recruitment of specific myeloid cells, and results in endothelial proliferation/leakiness. To test this hypothesis, we propose to: 1) define the relationship between Ras-activating mutations (RAM) and MPE formation; 2) identify tumor cell Ras-dependent signalling pathways and gene expression signature critical for MPE formation; 3) investigate the host response to tumor cells with RAM that results in MPE; and 4) target Ras and dependent signalling pathways as potential therapy for MPE. Studies will be performed using delivery of mouse/human tumors with/without RAM into the pleura of syngeneic/immunocompromized mice and are likely to yield new insights into the mechanisms of pleural tumor progression and to identify novel approaches to treatment of cancer patients with MPE.

SummaryHepatocellular carcinoma (HCC) is caused by chronic hepatitis and is the third most common cause of cancer-related death worldwide, with a rising incidence in first world countries. To date no effective therapies other than liver transplantation are available for this disease.
Previous studies have provided evidence that inflammatory signalling pathways (e.g. the NF-b pathway) are crucial modulators of liver cancer development. However, the exact mechanisms driving hepatitis-induced liver damage and cancer formation remain elusive. Among others, aberrant expression of cytotoxic cytokines is thought to be critically involved.
We have recently shown that the inflammatory cytokines lymphotoxin (LT)  and  are specifically upregulated in livers of patients suffering from hepatitis C and B virus-induced liver inflammation or HCC and that liver specific expression of LT and  in mice (AlbLT) suffices to induce inflammation-induced liver cancer development. We could further demonstrate that this depended on the presence of functional lymphocytes.
My proposal is pillared by three main approaches that all aim to elucidate the exact cellular and molecular mechanisms underlying chronic liver damage and HCC development in humans as well as in mouse models of inflammation- or carcinogen-induced liver cancer.
First, we will identify the particular immune cell type(s) (e.g. B- or T-lymphocytes; macrophages; NK-T cells) involved in HCC development. Although inflammatory signalling and immune cells appear to be important in HCC development it remains elusive, which immune cell type(s) contribute to inflammation induced liver cancer development.
Secondly, we will investigate how inflammatory signalling pathways induce chromosomal aberrations. It is known that inflammatory signalling cascades cause chromosomal aberrations; however, the detailed mechanisms by which this occurs are not fully understood. Additionally, we will determine how inflammatory signalling influences the pathways involved in DNA repair, replication and chromosomal segregation culminating in chromosomal aberrations and cancer.
Finally, we will examine the role of oval cells in liver cancer formation. Oval cells, which are putative liver-cancer stem cells, differentiate into either hepatocytes or cholangiocytes, proliferate under inflammatory conditions, and are found within HCC. However, their exact functional role in liver carcinogenesis is unknown. We will biochemically characterize proliferating and HCC-associated ovals cells in mouse models of inflammation-induced HCC and in diseased human liver tissues. This will pave the way for the development of the first genetic tools to deplete or express genes in an oval cell-specific manner.
The new scientific knowledge gained by these studies investigating how immune cells and inflammatory signalling induce chronic liver damage and cancer on a mechanistic level, and how oval cells contribute to HCC will provide the basis for future novel pharmacological approaches to treating inflammatory liver diseases and HCC in humans.

Hepatocellular carcinoma (HCC) is caused by chronic hepatitis and is the third most common cause of cancer-related death worldwide, with a rising incidence in first world countries. To date no effective therapies other than liver transplantation are available for this disease.
Previous studies have provided evidence that inflammatory signalling pathways (e.g. the NF-b pathway) are crucial modulators of liver cancer development. However, the exact mechanisms driving hepatitis-induced liver damage and cancer formation remain elusive. Among others, aberrant expression of cytotoxic cytokines is thought to be critically involved.
We have recently shown that the inflammatory cytokines lymphotoxin (LT)  and  are specifically upregulated in livers of patients suffering from hepatitis C and B virus-induced liver inflammation or HCC and that liver specific expression of LT and  in mice (AlbLT) suffices to induce inflammation-induced liver cancer development. We could further demonstrate that this depended on the presence of functional lymphocytes.
My proposal is pillared by three main approaches that all aim to elucidate the exact cellular and molecular mechanisms underlying chronic liver damage and HCC development in humans as well as in mouse models of inflammation- or carcinogen-induced liver cancer.
First, we will identify the particular immune cell type(s) (e.g. B- or T-lymphocytes; macrophages; NK-T cells) involved in HCC development. Although inflammatory signalling and immune cells appear to be important in HCC development it remains elusive, which immune cell type(s) contribute to inflammation induced liver cancer development.
Secondly, we will investigate how inflammatory signalling pathways induce chromosomal aberrations. It is known that inflammatory signalling cascades cause chromosomal aberrations; however, the detailed mechanisms by which this occurs are not fully understood. Additionally, we will determine how inflammatory signalling influences the pathways involved in DNA repair, replication and chromosomal segregation culminating in chromosomal aberrations and cancer.
Finally, we will examine the role of oval cells in liver cancer formation. Oval cells, which are putative liver-cancer stem cells, differentiate into either hepatocytes or cholangiocytes, proliferate under inflammatory conditions, and are found within HCC. However, their exact functional role in liver carcinogenesis is unknown. We will biochemically characterize proliferating and HCC-associated ovals cells in mouse models of inflammation-induced HCC and in diseased human liver tissues. This will pave the way for the development of the first genetic tools to deplete or express genes in an oval cell-specific manner.
The new scientific knowledge gained by these studies investigating how immune cells and inflammatory signalling induce chronic liver damage and cancer on a mechanistic level, and how oval cells contribute to HCC will provide the basis for future novel pharmacological approaches to treating inflammatory liver diseases and HCC in humans.

Max ERC Funding

1 212 190 €

Duration

Start date: 2010-10-01, End date: 2015-09-30

Project acronymMCTRinIA

ProjectResolution Pharmacology and Physiology of MCTR in Arthritis

Researcher (PI)Jesmond Dalli

Host Institution (HI)QUEEN MARY UNIVERSITY OF LONDON

Call DetailsStarting Grant (StG), LS4, ERC-2015-STG

SummaryChronic inflammation may result from failure of the host response to engage pro-resolving pathways. The current treatment armamentarium for chronic inflammatory conditions may lead to immune suppression. Thus, identification of novel therapeutics that control inflammation without immune suppression will provide an attractive alternative approach. This is especially important since incidence of these conditions increases with an ageing global population. In planaria, mice, human peripheral blood and milk I recently uncovered a new family of endogenous molecules, named Maresin Conjugates in Tissue Regeneration (MCTR). These potently regulate white blood cell responses, promote the resolution of acute inflammation and accelerate tissue regeneration. The aim of this Starting Grant is to identify pathways that lead to failed resolution in inflammatory arthritis, as a prototypical chronic inflammatory condition. The hypothesis is that MCTR biosynthesis is dysregulated in inflammatory arthritis, leading to an unbridled host response, chronic inflammation and tissue destruction. This proposal will employ a multipronged approach to test this hypothesis by 1) Determining MCTR regulation in self-resolving and delayed-resolving arthritis; 2) Investigating the host protective and tissue regenerative actions of MCTRs in inflammatory arthritis; 3) Establishing the MCTR biosynthetic pathway and 4) Determining the regulation if its components during self-limited and delayed-resolving arthritis. Anticipated results will uncover novel pathways that become dysregulated during failed resolution. Results from this Starting Grant will also identify targets and new therapeutic approaches that will engage pro-resolution programs as well as tissue regeneration in conditions characterised by persistent inflammation and hence failed resolution. This will lay the basis for informed structure-activity based studies and the design of therapeutics for treatment of chronic inflammatory conditions.

Chronic inflammation may result from failure of the host response to engage pro-resolving pathways. The current treatment armamentarium for chronic inflammatory conditions may lead to immune suppression. Thus, identification of novel therapeutics that control inflammation without immune suppression will provide an attractive alternative approach. This is especially important since incidence of these conditions increases with an ageing global population. In planaria, mice, human peripheral blood and milk I recently uncovered a new family of endogenous molecules, named Maresin Conjugates in Tissue Regeneration (MCTR). These potently regulate white blood cell responses, promote the resolution of acute inflammation and accelerate tissue regeneration. The aim of this Starting Grant is to identify pathways that lead to failed resolution in inflammatory arthritis, as a prototypical chronic inflammatory condition. The hypothesis is that MCTR biosynthesis is dysregulated in inflammatory arthritis, leading to an unbridled host response, chronic inflammation and tissue destruction. This proposal will employ a multipronged approach to test this hypothesis by 1) Determining MCTR regulation in self-resolving and delayed-resolving arthritis; 2) Investigating the host protective and tissue regenerative actions of MCTRs in inflammatory arthritis; 3) Establishing the MCTR biosynthetic pathway and 4) Determining the regulation if its components during self-limited and delayed-resolving arthritis. Anticipated results will uncover novel pathways that become dysregulated during failed resolution. Results from this Starting Grant will also identify targets and new therapeutic approaches that will engage pro-resolution programs as well as tissue regeneration in conditions characterised by persistent inflammation and hence failed resolution. This will lay the basis for informed structure-activity based studies and the design of therapeutics for treatment of chronic inflammatory conditions.

Max ERC Funding

1 964 303 €

Duration

Start date: 2016-03-01, End date: 2021-02-28

Project acronymMUCOSAL ER STRESS

ProjectXBP1 and Endoplasmic Reticulum Stress in Mucosal Homeostasis

Researcher (PI)Arthur Kaser

Host Institution (HI)THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE

Call DetailsStarting Grant (StG), LS4, ERC-2010-StG_20091118

SummaryEndoplasmic reticulum (ER) stress allows cells to cope with misfolded proteins through the Unfolded Protein Response (UPR). We have recently reported that unabated ER stress, a consequence of genetic deletion of X-box binding protein-1 (XBP1), in intestinal epithelial cells (IECs) leads to intestinal inflammation reminiscent of inflammatory bowel disease (IBD), and polymorphisms in XBP1 were associated with Crohn s disease and ulcerative colitis.
The current proposal is based on this central discovery, and rests on three major pillars. 1. Elucidation of the molecular pathways that connect unabated ER stress with inflammation, with the potential to identify novel therapeutics. 2. Testing the hypothesis that XBP1 deficiency may regulate colorectal cancer development, both sporadic and inflammation-associated. 3. Addressing the hypothesis that XBP1 and ER stress may contribute to the molecular pathology of primary sclerosing cholangitis (PSC) via affecting cholangiocyte biology. Insight from these studies may have implications well beyond mucosal inflammation as ER stress mechanisms have been suggested to play a role in a wide variety of diseases.

Endoplasmic reticulum (ER) stress allows cells to cope with misfolded proteins through the Unfolded Protein Response (UPR). We have recently reported that unabated ER stress, a consequence of genetic deletion of X-box binding protein-1 (XBP1), in intestinal epithelial cells (IECs) leads to intestinal inflammation reminiscent of inflammatory bowel disease (IBD), and polymorphisms in XBP1 were associated with Crohn s disease and ulcerative colitis.
The current proposal is based on this central discovery, and rests on three major pillars. 1. Elucidation of the molecular pathways that connect unabated ER stress with inflammation, with the potential to identify novel therapeutics. 2. Testing the hypothesis that XBP1 deficiency may regulate colorectal cancer development, both sporadic and inflammation-associated. 3. Addressing the hypothesis that XBP1 and ER stress may contribute to the molecular pathology of primary sclerosing cholangitis (PSC) via affecting cholangiocyte biology. Insight from these studies may have implications well beyond mucosal inflammation as ER stress mechanisms have been suggested to play a role in a wide variety of diseases.

Max ERC Funding

1 500 000 €

Duration

Start date: 2011-01-01, End date: 2015-12-31

Project acronymNiche Fibrosis

ProjectIdentification of regulatory signals from vascular niche in alveolar regeneration and pulmonary fibrosis

Researcher (PI)Joo-Hyeon Lee

Host Institution (HI)THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE

Call DetailsStarting Grant (StG), LS4, ERC-2015-STG

SummaryPulmonary fibrosis is a multifaceted and fatal disease that includes damaged alveolar epithelial cells and disorganization of multiple stromal cells. Dysregulation of multicellular crosstalk between epithelial and stromal cells is likely to contribute to fibrosis. However, the precise way this tissue damage occurs is unknown. I hypothesize that impaired function of lung epithelial stem cell lead to alveolar epithelial damage in pulmonary fibrosis, and which may be caused by altered stromal/niche cells. Epithelial injury repair and regeneration in the adult lung is carried out by numerous epithelial stem/progenitor cells. Recently, I identified a crucial interaction between lung endothelial cells and lung stem cells during alveolar injury response, and demonstrated a new regulatory signalling pathway that operates in endothelial cells to support alveolar injury repair by driving alveolar lineage specification of stem cells. Importantly, introduction of endothelial-derived factors into the lung after fibrotic damage enhances alveolar regeneration and reduces pulmonary fibrosis.
Given these results and unique my background knowledge, I will bring a new concept of stem cell-niche interactions in alveolar injury repair and pulmonary fibrosis. Using both in vivo murine and organoid culture, as well as human lung organoid culture systems, I will define 1) whether and how the fibrotic response affects lung stem cells and 2) how lung stem cells are regulated by endothelial cells that may comprise their respective niches during injury repair. 3) The mechanisms involved in the normal and pathological regulation of lung stem cells will be elucidated by determining secreted factors and regulatory signals endothelial cells confer through paracrine and direct physical interaction with stem cells. Insights gained from these studies will accelerate the development of novel and selective therapeutic approaches that directly target stem cells or their niches in pulmonary fibrosis.

Pulmonary fibrosis is a multifaceted and fatal disease that includes damaged alveolar epithelial cells and disorganization of multiple stromal cells. Dysregulation of multicellular crosstalk between epithelial and stromal cells is likely to contribute to fibrosis. However, the precise way this tissue damage occurs is unknown. I hypothesize that impaired function of lung epithelial stem cell lead to alveolar epithelial damage in pulmonary fibrosis, and which may be caused by altered stromal/niche cells. Epithelial injury repair and regeneration in the adult lung is carried out by numerous epithelial stem/progenitor cells. Recently, I identified a crucial interaction between lung endothelial cells and lung stem cells during alveolar injury response, and demonstrated a new regulatory signalling pathway that operates in endothelial cells to support alveolar injury repair by driving alveolar lineage specification of stem cells. Importantly, introduction of endothelial-derived factors into the lung after fibrotic damage enhances alveolar regeneration and reduces pulmonary fibrosis.
Given these results and unique my background knowledge, I will bring a new concept of stem cell-niche interactions in alveolar injury repair and pulmonary fibrosis. Using both in vivo murine and organoid culture, as well as human lung organoid culture systems, I will define 1) whether and how the fibrotic response affects lung stem cells and 2) how lung stem cells are regulated by endothelial cells that may comprise their respective niches during injury repair. 3) The mechanisms involved in the normal and pathological regulation of lung stem cells will be elucidated by determining secreted factors and regulatory signals endothelial cells confer through paracrine and direct physical interaction with stem cells. Insights gained from these studies will accelerate the development of novel and selective therapeutic approaches that directly target stem cells or their niches in pulmonary fibrosis.

SummaryObesity is associated with increased risk for epithelial tumors such as hepatocellular carcinoma (HCC). It is not known, however, whether obesity increases the risk for HCC simply because it promotes cirrhosis, a general risk factor for HCC, or through other mechanisms that operate independently of cirrhosis. Among these, obesity is associated with a chronic inflammatory state, with the release of cytokines such as IL-6 and TNFalpha, well-known HCC mediators. Obesity is normally linked to diabetes and in consequence, to hyperinsulinemia. This increase in circulating insulin levels is suggested to be a factor that contributes to cancer. Moreover, the increase in free fatty acids (FFA) in blood among obese patients promotes a compensatory response from liver that activates the transcription of genes required for beta-oxidation, leading to a reduction in non-physiological stores of lipids in the liver. This increase in beta-oxidation could result in oxidative stress, inflammation and the production of lipid peroxidation bioproducts, which are known mutagens. The precise mechanisms whereby FFA and cytosolic triglycerides exert their effects, resulting in the diabetic phenotype, remain poorly understood. Emerging evidence nonetheless links microRNA (miRNA) with lipid metabolism, suggesting that these small RNAs mediate this increase in beta-oxidation.
Our goal is to study how the components of the obesity state (inflammation, steatosis hyperinsulinemia and microRNA control of gene regulation) affect HCC development. We will use several mouse models in which one or more of these factors are reduced following induction of metabolic disease. We will also determine whether specific miRNAs that are down- or upregulated in the liver of mice on a high fat diet are implicated in HCC development.

Obesity is associated with increased risk for epithelial tumors such as hepatocellular carcinoma (HCC). It is not known, however, whether obesity increases the risk for HCC simply because it promotes cirrhosis, a general risk factor for HCC, or through other mechanisms that operate independently of cirrhosis. Among these, obesity is associated with a chronic inflammatory state, with the release of cytokines such as IL-6 and TNFalpha, well-known HCC mediators. Obesity is normally linked to diabetes and in consequence, to hyperinsulinemia. This increase in circulating insulin levels is suggested to be a factor that contributes to cancer. Moreover, the increase in free fatty acids (FFA) in blood among obese patients promotes a compensatory response from liver that activates the transcription of genes required for beta-oxidation, leading to a reduction in non-physiological stores of lipids in the liver. This increase in beta-oxidation could result in oxidative stress, inflammation and the production of lipid peroxidation bioproducts, which are known mutagens. The precise mechanisms whereby FFA and cytosolic triglycerides exert their effects, resulting in the diabetic phenotype, remain poorly understood. Emerging evidence nonetheless links microRNA (miRNA) with lipid metabolism, suggesting that these small RNAs mediate this increase in beta-oxidation.
Our goal is to study how the components of the obesity state (inflammation, steatosis hyperinsulinemia and microRNA control of gene regulation) affect HCC development. We will use several mouse models in which one or more of these factors are reduced following induction of metabolic disease. We will also determine whether specific miRNAs that are down- or upregulated in the liver of mice on a high fat diet are implicated in HCC development.

Max ERC Funding

1 498 043 €

Duration

Start date: 2010-12-01, End date: 2016-11-30

Project acronymP73CANCER

Projectp73 dependence in cancer: from molecular mechanisms to therapeutic targeting

Researcher (PI)Thorsten Stiewe

Host Institution (HI)PHILIPPS UNIVERSITAET MARBURG

Call DetailsStarting Grant (StG), LS4, ERC-2010-StG_20091118

Summaryp73 is a transcription factor of the p53 tumor suppressor family. In approximately 50% of all cancer patients
the tumor suppressor function of p53 is irreversibly disabled by point mutations, which makes p53 one of the
most frequently mutated genes in cancer. This is entirely different for p73 and much of my previous work in
this field has been devoted to research on the role of p73 in cancer.
In sharp contrast to p53, p73 is often highly expressed in its wild-type form in solid tumors compared to
the surrounding normal tissue. This suggests the rather challenging hypothesis that p73 has oncogenic
functions in cancer cells, which promote tumor progression and therapy resistance. This concept is supported
by clinical data demonstrating p73 overexpression to be correlated with advanced tumor stage, metastasis,
therapy resistance and poor overall survival in multiple tumor entities including the ‘major killers’: breast,
lung and colorectal cancer. When p73 is depleted from cancer cell lines, tumor cell proliferation and
tumorigenicity are reduced, indicating that tumor cells with high p73 expression are p73-dependent. This
places p73 in line with oncogenes like Myc or mutant Ras, which are similarly essential for the tumorigenic
phenotype. However, tumor cells are not only addicted to a particular oncogene but in many cases codependent
on other cellular factors - a phenomenon termed ‘non-oncogene addiction’. Since p73 also has
tumor suppressive functions, p73-dependent tumor cells are likely to be critically dependent on cooperating
factors, which keep the proapoptotic and tumor suppressive functions of p73 in check. Inhibition of these
factors would be ‘synthetically lethal’ with overexpression of p73. From a clinical point-of-view it would be
extremely valuable, if we knew these factors and were able to block them in order to reactivate p73’s tumor
suppressor activity and trigger growth inhibition or cell death. This approach promises to be specifically
effective in the therapeutically challenging p73-dependent tumors with little or no side effects in normal
tissues with low p73 expression.
The goal of this project is therefore the identification and validation of such synthetic lethal interactions
with p73 using different functional genomics approaches. In the first part of the project we will characterize
the impact of p73-dependence on gene expression using genome-wide expression and global chromatin state
profiling. This will enhance our molecular understanding why cancer cells rely on high-level expression of
p73. In addition, this will pinpoint genes and pathways, which are co-expressed and activated together with
p73 and which are therefore candidate genes for therapeutic targeting of p73-dependent cancer cells. In the
second part of the project we will use RNAi screening techniques on a genome-wide scale to identify in an
unbiased manner cellular factors, which enable cancer cells to tolerate high-level expression of p73. These
genes are essential for long-term proliferation and survival in the context of p73 overexpression and could be
ideal drug targets for a tumor-selective therapy of the prognostically dismal class of p73-dependent tumors

p73 is a transcription factor of the p53 tumor suppressor family. In approximately 50% of all cancer patients
the tumor suppressor function of p53 is irreversibly disabled by point mutations, which makes p53 one of the
most frequently mutated genes in cancer. This is entirely different for p73 and much of my previous work in
this field has been devoted to research on the role of p73 in cancer.
In sharp contrast to p53, p73 is often highly expressed in its wild-type form in solid tumors compared to
the surrounding normal tissue. This suggests the rather challenging hypothesis that p73 has oncogenic
functions in cancer cells, which promote tumor progression and therapy resistance. This concept is supported
by clinical data demonstrating p73 overexpression to be correlated with advanced tumor stage, metastasis,
therapy resistance and poor overall survival in multiple tumor entities including the ‘major killers’: breast,
lung and colorectal cancer. When p73 is depleted from cancer cell lines, tumor cell proliferation and
tumorigenicity are reduced, indicating that tumor cells with high p73 expression are p73-dependent. This
places p73 in line with oncogenes like Myc or mutant Ras, which are similarly essential for the tumorigenic
phenotype. However, tumor cells are not only addicted to a particular oncogene but in many cases codependent
on other cellular factors - a phenomenon termed ‘non-oncogene addiction’. Since p73 also has
tumor suppressive functions, p73-dependent tumor cells are likely to be critically dependent on cooperating
factors, which keep the proapoptotic and tumor suppressive functions of p73 in check. Inhibition of these
factors would be ‘synthetically lethal’ with overexpression of p73. From a clinical point-of-view it would be
extremely valuable, if we knew these factors and were able to block them in order to reactivate p73’s tumor
suppressor activity and trigger growth inhibition or cell death. This approach promises to be specifically
effective in the therapeutically challenging p73-dependent tumors with little or no side effects in normal
tissues with low p73 expression.
The goal of this project is therefore the identification and validation of such synthetic lethal interactions
with p73 using different functional genomics approaches. In the first part of the project we will characterize
the impact of p73-dependence on gene expression using genome-wide expression and global chromatin state
profiling. This will enhance our molecular understanding why cancer cells rely on high-level expression of
p73. In addition, this will pinpoint genes and pathways, which are co-expressed and activated together with
p73 and which are therefore candidate genes for therapeutic targeting of p73-dependent cancer cells. In the
second part of the project we will use RNAi screening techniques on a genome-wide scale to identify in an
unbiased manner cellular factors, which enable cancer cells to tolerate high-level expression of p73. These
genes are essential for long-term proliferation and survival in the context of p73 overexpression and could be
ideal drug targets for a tumor-selective therapy of the prognostically dismal class of p73-dependent tumors

Max ERC Funding

1 499 040 €

Duration

Start date: 2010-10-01, End date: 2016-09-30

Project acronymPAPAstudy

ProjectPodocyte Adaptation Proliferation and Ageing

Researcher (PI)Guillaume Canaud

Host Institution (HI)UNIVERSITE PARIS DESCARTES

Call DetailsStarting Grant (StG), LS4, ERC-2015-STG

SummaryMy research project is focused on the biology of the podocyte during chronic kidney disease progression, one of the major public health challenges of the 21st Century. The podocyte is a highly specialized cell with foot processes expansion that function as vasculature-supporting cells, producing basement membrane components and a number of vascular growth factors. These cells play an essential role in renal physiology but their unique localization, as the primary filter, exposes them to many stresses in pathological conditions. In fact these cells are the direct target of many diseases such as diabetes or HIV. The molecular events and structural changes in podocyte during adaptation to nephron reduction are not well-characterized and efficient therapeutics dramatically lacking. In order to achieve the ultimate goal of improving care provided to patients suffering from renal failure, the dissection of the molecular pathways involved in podocyte adaptation and deterioration processes is mandatory. This project will combine complementary in vivo and in vitro approaches, experimental models of chronic kidney disease on genetically modified mice, as well as the use of a very innovative and new technology of bioengineering to explore the molecular and structural changes of the podocytes during kidney diseases. This work will be centralized on AKT2 and its partners, as we previously demonstrated its major role in podocyte adaptation to nephron reduction, but also on new players using unbiased approaches. In parallel and consistently with my previous works, we will systematically extend our findings to human through our unique Renal Biobank of Necker Hospital (Paris). The accomplishment of this project may lead to the discovery of novel therapeutic targets and strategies to slow down the progression of kidney disease.

My research project is focused on the biology of the podocyte during chronic kidney disease progression, one of the major public health challenges of the 21st Century. The podocyte is a highly specialized cell with foot processes expansion that function as vasculature-supporting cells, producing basement membrane components and a number of vascular growth factors. These cells play an essential role in renal physiology but their unique localization, as the primary filter, exposes them to many stresses in pathological conditions. In fact these cells are the direct target of many diseases such as diabetes or HIV. The molecular events and structural changes in podocyte during adaptation to nephron reduction are not well-characterized and efficient therapeutics dramatically lacking. In order to achieve the ultimate goal of improving care provided to patients suffering from renal failure, the dissection of the molecular pathways involved in podocyte adaptation and deterioration processes is mandatory. This project will combine complementary in vivo and in vitro approaches, experimental models of chronic kidney disease on genetically modified mice, as well as the use of a very innovative and new technology of bioengineering to explore the molecular and structural changes of the podocytes during kidney diseases. This work will be centralized on AKT2 and its partners, as we previously demonstrated its major role in podocyte adaptation to nephron reduction, but also on new players using unbiased approaches. In parallel and consistently with my previous works, we will systematically extend our findings to human through our unique Renal Biobank of Necker Hospital (Paris). The accomplishment of this project may lead to the discovery of novel therapeutic targets and strategies to slow down the progression of kidney disease.

Max ERC Funding

1 479 260 €

Duration

Start date: 2016-03-01, End date: 2021-02-28

Project acronymSIADIA

ProjectSiglecs as mediators of the pancreatic cellular crosstalk in diabetes

Researcher (PI)Kathrin Ulrike Maedler

Host Institution (HI)UNIVERSITAET BREMEN

Call DetailsStarting Grant (StG), LS4, ERC-2010-StG_20091118

SummaryThe mechanisms of the immune and endocrine cell interaction within the islet and resulting β-cell death are
highly complex and largely unknown. To investigate the cellular crosstalk in the pancreas and how its
disturbance leads to insufficient insulin production is important to understand the pathology of the disease. This
is the major goal of this project.
Activation of inflammation is not only a trigger for β-cell destruction, but also a major factor for the metabolic
syndrome, including insulin resistance and complications of diabetes.
Signalling and activation of immune cells is facilitated by secreted pro-inflammatory stimulators and via cell-cell
interactions. I propose that a group of adhesion and signalling molecules, the Siglecs (sialic acid–binding
immunoglobulin (Ig)-like lectins) mediate such interactions. They are responsible for immune system activation
and have been initially found in cells of hematopoietic origin. I made the groundbreaking observation of cell
type specific Siglec expression in the human pancreas: Siglecs were differentially expressed in glucagon
producing α-cells, and in insulin producing β-cells. A diabetic milieu had an inductive effect on Siglec
expression in the α-cells, but lead to decreased β-cell specific Siglecs. This loss of Siglecs in the β-cell could be
detrimental and result in an excessive cytokine release and in turn switches on Siglec responses in neighbouring
cells. In my proposed studies I will investigate the role of Siglecs in the cellular network in islets and in the
circulation to probe whether changes in Siglec expression are causative in the development of diabetes.
My project is a pioneer and multidisciplinary study combining the current knowledge of glycobiochemistry
and β-cell biology in diabetes. The project uses multi-model cell systems of healthy and diseased human
pancreatic tissue, isolated human islets, isolated human β-cells as well as diabetic mouse models, all of them
being absolutely novel and high-risk to a large extend.

The mechanisms of the immune and endocrine cell interaction within the islet and resulting β-cell death are
highly complex and largely unknown. To investigate the cellular crosstalk in the pancreas and how its
disturbance leads to insufficient insulin production is important to understand the pathology of the disease. This
is the major goal of this project.
Activation of inflammation is not only a trigger for β-cell destruction, but also a major factor for the metabolic
syndrome, including insulin resistance and complications of diabetes.
Signalling and activation of immune cells is facilitated by secreted pro-inflammatory stimulators and via cell-cell
interactions. I propose that a group of adhesion and signalling molecules, the Siglecs (sialic acid–binding
immunoglobulin (Ig)-like lectins) mediate such interactions. They are responsible for immune system activation
and have been initially found in cells of hematopoietic origin. I made the groundbreaking observation of cell
type specific Siglec expression in the human pancreas: Siglecs were differentially expressed in glucagon
producing α-cells, and in insulin producing β-cells. A diabetic milieu had an inductive effect on Siglec
expression in the α-cells, but lead to decreased β-cell specific Siglecs. This loss of Siglecs in the β-cell could be
detrimental and result in an excessive cytokine release and in turn switches on Siglec responses in neighbouring
cells. In my proposed studies I will investigate the role of Siglecs in the cellular network in islets and in the
circulation to probe whether changes in Siglec expression are causative in the development of diabetes.
My project is a pioneer and multidisciplinary study combining the current knowledge of glycobiochemistry
and β-cell biology in diabetes. The project uses multi-model cell systems of healthy and diseased human
pancreatic tissue, isolated human islets, isolated human β-cells as well as diabetic mouse models, all of them
being absolutely novel and high-risk to a large extend.

Max ERC Funding

1 363 847 €

Duration

Start date: 2011-01-01, End date: 2015-12-31

Project acronymSiCMetabol

ProjectSignaling Cascades in Metabolic Diseases

Researcher (PI)Grzegorz Piotr Sumara

Host Institution (HI)JULIUS-MAXIMILIANS-UNIVERSITAT WURZBURG

Call DetailsStarting Grant (StG), LS4, ERC-2015-STG

SummaryOver 380 million people suffer from diabetes worldwide, with majority of cases being attributed to type 2 diabetes (T2D). Obesity is a major risk factor predisposing to the development of this disease. T2D is characterized by peripheral insulin resistance in combination with relative insulin deficiency that results in hyperglycemia and hyperlipidemia. Liver and adipose tissue are central for regulation of glucose and lipids levels. However, during T2D the hepatic glucose uptake is reduced while rates of gluconeogenesis and lipogenesis are increased. In the adipose tissue, T2D leads to decreased glucose uptake, perturbations in secretion of adipokines and increased lipolysis. Importantly, dysfunction of the liver and the adipose tissue during T2D is caused by defective phosphorylation signaling cascades and normalization of these pathways was shown to attenuate the course of T2D. However, the specific roles of different classes of signaling molecules in these organs remain poorly characterized. We hypothesize that the cross-talk of different classes of signaling molecules determines regulation of metabolism.
Thus, we aim to identify the signaling networks regulating metabolism. The results generated in my own laboratory suggest that the Pkd family kinases are the crucial regulators of metabolic homeostasis. Specifically, Pkd1 and Pkd2 promote obesity and diabetes while Pkd3 controls liver function. Thus, we plan to characterize the molecular mechanisms controlling Pkds signaling. In parallel, we will utilize screening approaches to identify novel, non-canonical signaling modules (phosphatases and components of the ubiquitin system) regulating abundance, localization and phosphorylation of targets of Pkds and, in the long term, also other kinases implicated in T2D.
By identifying and characterizing the essential signaling networks in liver and adipose tissue the project will contribute to more targeted pharmacological strategies for the treatment of T2D.

Over 380 million people suffer from diabetes worldwide, with majority of cases being attributed to type 2 diabetes (T2D). Obesity is a major risk factor predisposing to the development of this disease. T2D is characterized by peripheral insulin resistance in combination with relative insulin deficiency that results in hyperglycemia and hyperlipidemia. Liver and adipose tissue are central for regulation of glucose and lipids levels. However, during T2D the hepatic glucose uptake is reduced while rates of gluconeogenesis and lipogenesis are increased. In the adipose tissue, T2D leads to decreased glucose uptake, perturbations in secretion of adipokines and increased lipolysis. Importantly, dysfunction of the liver and the adipose tissue during T2D is caused by defective phosphorylation signaling cascades and normalization of these pathways was shown to attenuate the course of T2D. However, the specific roles of different classes of signaling molecules in these organs remain poorly characterized. We hypothesize that the cross-talk of different classes of signaling molecules determines regulation of metabolism.
Thus, we aim to identify the signaling networks regulating metabolism. The results generated in my own laboratory suggest that the Pkd family kinases are the crucial regulators of metabolic homeostasis. Specifically, Pkd1 and Pkd2 promote obesity and diabetes while Pkd3 controls liver function. Thus, we plan to characterize the molecular mechanisms controlling Pkds signaling. In parallel, we will utilize screening approaches to identify novel, non-canonical signaling modules (phosphatases and components of the ubiquitin system) regulating abundance, localization and phosphorylation of targets of Pkds and, in the long term, also other kinases implicated in T2D.
By identifying and characterizing the essential signaling networks in liver and adipose tissue the project will contribute to more targeted pharmacological strategies for the treatment of T2D.

Max ERC Funding

1 499 128 €

Duration

Start date: 2016-06-01, End date: 2021-05-31

Project acronymStemProteostasis

ProjectMediation of stem cell identity and aging by proteostasis

Researcher (PI)David Vilchez Guerrero

Host Institution (HI)UNIVERSITAET ZU KOELN

Call DetailsStarting Grant (StG), LS4, ERC-2015-STG

SummaryBy 2050, the global population over the age of 80 will triple. Thus, research for improving the quality of life at older age can be of enormous benefit for our ever-aging society. To address this challenge we propose an innovative approach based on a combination of stem cell research with genetic experiments in C. elegans. Mechanisms that promote protein homeostasis (proteostasis) slow down aging and decrease the incidence of age-related diseases. Since human embryonic stem cells (hESCs) replicate continuously in the absence of senescence, we hypothesize that they can provide a novel paradigm to study proteostasis and its demise in aging. We have found that hESCs exhibit increased proteasome activity. Moreover, we have uncovered that the proteasome subunit RPN-6 is required for this activity and sufficient to extend healtshpan in C. elegans. However, the mechanisms by which the proteasome regulates hESC function remain unknown. Our first aim is to define how the proteasome regulates not only hESC identity but also aging and the onset of age-related diseases. Moreover, one of the next challenges is to define how other proteostasis pathways impinge upon hESC function. We hypothesize that, in addition to the proteasome, hESCs differentially regulate other subcellular stress response pathways designed to protect them from disequilibrium in the folding and degradation of their proteome. We will perform a comprehensive study of proteostasis of hESCs and mimic this network in somatic cells to alleviate age-related diseases. Finally, we will determine whether loss of proteostasis promotes somatic stem cell (SC) exhaustion, which is one of the most obvious characteristics of the aging process and contributes to tissue degeneration. By using mouse models we will examine whether sustained proteostasis delays neural SC exhaustion. Our research will have an impact in several fields such as stem cell research, neurogenesis, proteostasis, aging and age-related diseases.

By 2050, the global population over the age of 80 will triple. Thus, research for improving the quality of life at older age can be of enormous benefit for our ever-aging society. To address this challenge we propose an innovative approach based on a combination of stem cell research with genetic experiments in C. elegans. Mechanisms that promote protein homeostasis (proteostasis) slow down aging and decrease the incidence of age-related diseases. Since human embryonic stem cells (hESCs) replicate continuously in the absence of senescence, we hypothesize that they can provide a novel paradigm to study proteostasis and its demise in aging. We have found that hESCs exhibit increased proteasome activity. Moreover, we have uncovered that the proteasome subunit RPN-6 is required for this activity and sufficient to extend healtshpan in C. elegans. However, the mechanisms by which the proteasome regulates hESC function remain unknown. Our first aim is to define how the proteasome regulates not only hESC identity but also aging and the onset of age-related diseases. Moreover, one of the next challenges is to define how other proteostasis pathways impinge upon hESC function. We hypothesize that, in addition to the proteasome, hESCs differentially regulate other subcellular stress response pathways designed to protect them from disequilibrium in the folding and degradation of their proteome. We will perform a comprehensive study of proteostasis of hESCs and mimic this network in somatic cells to alleviate age-related diseases. Finally, we will determine whether loss of proteostasis promotes somatic stem cell (SC) exhaustion, which is one of the most obvious characteristics of the aging process and contributes to tissue degeneration. By using mouse models we will examine whether sustained proteostasis delays neural SC exhaustion. Our research will have an impact in several fields such as stem cell research, neurogenesis, proteostasis, aging and age-related diseases.

SummaryChronic lung diseases including COPD and cancer account for more than 10% of deaths in European countries and lung cancer remains the most common and most lethal cancer type worldwide. Lung cancer mortality rates have also remained constant for over 40 years, largely due to late stage diagnosis and frequent metastases. Despite these trends the signalling pathways regulating lung cancers remain largely unexplored. Although loss of Tumour Suppressor of Lung Cancer 1 (TSLC1) is implicated in lung cancer metastases its normal lung functions remain entirely unknown. Recently, I determined that the TSLC1 signalling pathway regulates cell proliferation, motility, skin repair, and cancer severity by inhibiting stem cell EMTs. This proposal will determine how TSLC1 regulates lung EMTs to influence repair and disease outcomes. I will correlate TSLC1 expression with EMTs in repairing and diseased lungs, investigate how lung TSLC1
signalling influences repair and disease outcomes using transgenic and knockout mice, and identify mechanisms through which TSLC1 modulates lung phenotypes using in vitro overexpression and knockdown studies. This research will elucidate TSLC1 signalling pathway functions in lungs and should suggest new therapeutic opportunities for human lung disease.

Chronic lung diseases including COPD and cancer account for more than 10% of deaths in European countries and lung cancer remains the most common and most lethal cancer type worldwide. Lung cancer mortality rates have also remained constant for over 40 years, largely due to late stage diagnosis and frequent metastases. Despite these trends the signalling pathways regulating lung cancers remain largely unexplored. Although loss of Tumour Suppressor of Lung Cancer 1 (TSLC1) is implicated in lung cancer metastases its normal lung functions remain entirely unknown. Recently, I determined that the TSLC1 signalling pathway regulates cell proliferation, motility, skin repair, and cancer severity by inhibiting stem cell EMTs. This proposal will determine how TSLC1 regulates lung EMTs to influence repair and disease outcomes. I will correlate TSLC1 expression with EMTs in repairing and diseased lungs, investigate how lung TSLC1
signalling influences repair and disease outcomes using transgenic and knockout mice, and identify mechanisms through which TSLC1 modulates lung phenotypes using in vitro overexpression and knockdown studies. This research will elucidate TSLC1 signalling pathway functions in lungs and should suggest new therapeutic opportunities for human lung disease.

SummaryThere is increasing evidence that the body s ability to mount an immune response to cancer cells may dictate which patients are cured of cancer by conventional therapy. Macrophages have a central role in both innate and adaptive immunity; both human and experimental cancers become infiltrated by macrophages, however tumour-associated macrophages (TAM) are corrupted by the tumour microenvironment to promote survival, invasion and metastasis of cancer cells. TAM also contribute to immune-suppression in cancer and evasion of anti-tumour immunity. It is not clear what factors promote the pro-tumour TAM phenotype or the signalling pathways involved. The intrinsic anti-tumour potential of macrophages as innate immune cells and their abundance in solid tumours makes them an attractive therapeutic target. The challenge is to block the cancer-promoting function of these cells and restore their anti-tumour effects. We have previously shown that NF-ºB inhibits the classical activation of macrophages in the context of inflammation and cancer and our preliminary data show that NF-ºB activation in TAM inhibits anti-tumour immunity in transplantable models of cancer. Further studies have shown a subset of IKK²-regulated genes in macrophages are dependent on p38 MAP Kinase (MAPK14) and targeting p38 activity in macrophages also blocks the TAM phenotype. These data suggest IKK²-p38 signalling maintains the pro-tumour phenotype of TAM and inhibits anti-tumour immunity.In this project we will extend our preliminary observations in transplantable tumour models to clinically relevant genetic models of spontaneous cancer in mice. We will also map IKK² and p38 target genes in TAM and investigate IKK²/p38 dependent mechanisms of gene regulation.

There is increasing evidence that the body s ability to mount an immune response to cancer cells may dictate which patients are cured of cancer by conventional therapy. Macrophages have a central role in both innate and adaptive immunity; both human and experimental cancers become infiltrated by macrophages, however tumour-associated macrophages (TAM) are corrupted by the tumour microenvironment to promote survival, invasion and metastasis of cancer cells. TAM also contribute to immune-suppression in cancer and evasion of anti-tumour immunity. It is not clear what factors promote the pro-tumour TAM phenotype or the signalling pathways involved. The intrinsic anti-tumour potential of macrophages as innate immune cells and their abundance in solid tumours makes them an attractive therapeutic target. The challenge is to block the cancer-promoting function of these cells and restore their anti-tumour effects. We have previously shown that NF-ºB inhibits the classical activation of macrophages in the context of inflammation and cancer and our preliminary data show that NF-ºB activation in TAM inhibits anti-tumour immunity in transplantable models of cancer. Further studies have shown a subset of IKK²-regulated genes in macrophages are dependent on p38 MAP Kinase (MAPK14) and targeting p38 activity in macrophages also blocks the TAM phenotype. These data suggest IKK²-p38 signalling maintains the pro-tumour phenotype of TAM and inhibits anti-tumour immunity.In this project we will extend our preliminary observations in transplantable tumour models to clinically relevant genetic models of spontaneous cancer in mice. We will also map IKK² and p38 target genes in TAM and investigate IKK²/p38 dependent mechanisms of gene regulation.

SummaryObesity and T2D affect large populations and cause a decline in life expectancy if untreated. The pandemic proportion of obesity and inaptitude of anti-obesity approaches reflect our limited understanding of its complex environmental and genetic etiology. Genome-wide association studies revealed that disease-associated risk variants are often situated in those 98% of the genome not encoding for proteins. This noncoding genomic space yet does not reflect ‘Junk DNA’ but gives rise to >10,000 noncoding RNAs like microRNAs and long, noncoding RNAs (lncRNAs) that implicated in control of glucose metabolism and energy homeostasis also by the applicant (Kornfeld et al. Nature 2013).
LncRNAs were paraphrased as 'Dark matter of the genome' due to their tissue-specific and dynamic expression that contrast their poorly understood role in gene regulation. In the 1st part of this proposal, we ask if lncRNAs regulate glucose metabolism and are involved in the obesity-associated dysregulation of insulin signaling in the liver, the major glucoregulatory organ in mammals. Using RNA-Seq and novel lncRNA prediction algorithms, we observed that obesity alters expression of 28 annotated and 15 hitherto unknown lncRNAs in two mouse models of obesity. To identify lncRNAs causally controlling glucose metabolism, we established a siRNA screening system that allows functional interrogation of >650 lncRNAs. These in vitro findings serve as entry for the generation of lncRNA knockout mice that are metabolically phenotyped. In the 2nd part, we hypothesize that germline ncRNAs could control the transgenerational consequences of paternal obesity as shown for lower organisms. This builds upon unpublished findings from our lab showing that obesity profoundly changes expression of germline ncRNAs. In-vitro fertilization and intergenerational breedings will trace the legacy of paternal obesity across generations and reveal ncRNAs involved in this ‘Lamarckian’ control of glucose metabolism.

Obesity and T2D affect large populations and cause a decline in life expectancy if untreated. The pandemic proportion of obesity and inaptitude of anti-obesity approaches reflect our limited understanding of its complex environmental and genetic etiology. Genome-wide association studies revealed that disease-associated risk variants are often situated in those 98% of the genome not encoding for proteins. This noncoding genomic space yet does not reflect ‘Junk DNA’ but gives rise to >10,000 noncoding RNAs like microRNAs and long, noncoding RNAs (lncRNAs) that implicated in control of glucose metabolism and energy homeostasis also by the applicant (Kornfeld et al. Nature 2013).
LncRNAs were paraphrased as 'Dark matter of the genome' due to their tissue-specific and dynamic expression that contrast their poorly understood role in gene regulation. In the 1st part of this proposal, we ask if lncRNAs regulate glucose metabolism and are involved in the obesity-associated dysregulation of insulin signaling in the liver, the major glucoregulatory organ in mammals. Using RNA-Seq and novel lncRNA prediction algorithms, we observed that obesity alters expression of 28 annotated and 15 hitherto unknown lncRNAs in two mouse models of obesity. To identify lncRNAs causally controlling glucose metabolism, we established a siRNA screening system that allows functional interrogation of >650 lncRNAs. These in vitro findings serve as entry for the generation of lncRNA knockout mice that are metabolically phenotyped. In the 2nd part, we hypothesize that germline ncRNAs could control the transgenerational consequences of paternal obesity as shown for lower organisms. This builds upon unpublished findings from our lab showing that obesity profoundly changes expression of germline ncRNAs. In-vitro fertilization and intergenerational breedings will trace the legacy of paternal obesity across generations and reveal ncRNAs involved in this ‘Lamarckian’ control of glucose metabolism.